Crystalline 2-O-α-D-glucopyranosyl-L-ascorbic acid, and its preparation and uses

ABSTRACT

A method is provided for purifying 2-0-alpha-D-glucopyranosyl-L-ascorbic acid. The method comprises: providing a mixture containing 2-0-alpha-D-glucopyranosyl-L-ascorbic acid together with a water-soluble contaminant; and separating said mixture using a water-insoluble carrier in accordance with molecular weight or affinity into at least two fractions. The fractions comprise:   (i) a first fraction which is rich in 2-0-alpha-D-glucopyranosyl-L-ascorbic acid, and (ii) a second fraction which is rich in the contaminant. The first fraction is recovered.

This is a division of application Ser. No. 07/501,900 filed Mar. 30,1990, pending.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a novel substance, a crystalline2-O-α-D-glucopyranosyl-L-ascorbic acid, and its preparation and uses.

2. Description of the Prior Art

L-Ascorbic acid, which has the chemical structure shown by the formula[I]: ##STR1## is not synthesized in vivo in human, monkey and guineapig, and therefore is listed as an essential nutritive element, i.e.vitamin C.

L-Ascorbic acid takes part in some physiological activities in vivo; forexample, in the hydroxylation of proline and lysine which are necessaryto synthesize collagen as the main element of living connective tissues;the oxidation-reduction reaction of cytochrome C wherein Fe⁺⁺⁺ isreduced into Fe⁺⁺ ; and in immunopotentiation via the increase ofleukocytes. Thus vitamin C plays a significant role in the maintenanceand promotion of health in living body.

Scurvy has been known long as a condition due to a deficiency ofL-ascorbic acid, and is marked by weakness of the skin, petechialhemorrhage, ecchymosis, and hemorrhages in the gingiva and marrow. Toprevent scurvy for the maintenance of health, a recommended dailyadministration (RDA) is established for L-ascorbic acid; in particular,60 mg for adult males and 50 mg for adult females.

Nowadays the use of L-ascorbic acid is not limited to agents whichenrich vitamin C as an essential nutritive element, but is extending tovarious applications. More particularly, because of its chemicalstructure and physiological activities, L-ascorbic acid is useful as asouring agent, reductant, antioxidant, bleaching agent and stabilizer invarious chemical reagents, foods and beverages; in pharmaceuticalsagainst susceptive diseases such as prevention and treatment for viraldiseases, bacterial diseases and malignant tumors; and further as areductant, uv-absorbent and melanin-formation inhibitor in cosmeticsincluding skin-refining agent and skin-whitening agent.

The major drawback of L-ascorbic acid is that it readily loses itsphysiological activities because of its direct reducing activity, poorstability and high susceptibility to oxidation.

To stabilize L-ascorbic acid, some saccharide derivatives of L-ascorbicacid have been proposed. For example, we disclosed in Vitamin, Vol. 43,pp. 205-209 (1971), ibid., Vol. 47, pp. 259-267 (1973), and JapanesePatent Publication No. 38,158/73 a biochemical synthesis of L-ascorbicacid glucosides.

Because of the facts that the glucosides are prepared by similarmethods; that the formation of an ether bond at the primary alcoholgroup which is located at the number six carbon atom in L-ascorbic acidleads to the glucosides as described in the Japanese Patent Publication,for example, on the 2nd column, lines 14-16; that thesaccharide-transfer reaction from maltose to an α-glucosyl group isresponsible for the formation of glucosides; and that the glucosidesexhibit a direct reducing activity, their chemical structure would beshown by the formula [II]: ##STR2##

As obvious from the results in the Japanese Patent Publication, thetable in Example 1, the stability of the glucosides is superior to thatof L-ascorbic acid, but is not enough for their commercialization.

Ishido et al. disclose in Japanese Patent Publication No. 5,920/83 anorganic chemical process to synthesize saccharide derivatives ofL-ascorbic acid.

These derivatives are, however, those wherein all the D-glucoses arebound in the β-fashion because up to 21 β-D-glucopyranosyl typederivatives of L-ascorbic acid including2,3-di-O-(β-D-glucopyranosyl)-L-ascorbic acid are listed for explanationon the 7th column, line 6 to the 8th column, line 11.

Masamoto et al. disclose in Japanese Patent Publication No. 198,498/83an organic chemical process to synthesize saccharide derivatives ofL-ascorbic acid which are also of the β-glucosyl type.

Studies on the β-D-glucopyranosyl type derivatives of L-ascorbic acidconfirmed that they hardly exhibit desired physiological activities inliving body, especially in humans. Furthermore, conventional organicchemical processes have the drawbacks that they are inferior ineconomical efficiency because the reaction is very complicated and lowin yield, and the establishment of non-toxicity and safeness for theresultant derivatives is very difficult.

As described above, the proposals of saccharide derivatives ofL-ascorbic acid in the prior art have proved unsatisfactory in view ofstability, safeness, physiological activity and economical efficiency,and not been practiced hitherto.

The present invention has as an object to overcome the drawbacks ofconventional saccharide derivatives of L-ascorbic acid. Moreparticularly, we studied a novel saccharide derivative of L-ascorbicacid which is obtainable by a biochemical process utilizing asaccharide-transfer reaction.

As disclosed in the specification of Japanese Patent Application No.127,072/89, we discovered a novel substance, an α-glycosyl-L-ascorbicacid, especially, 2-O-α-D-glucopyranosyl-L-ascorbic acid, which is freefrom direct reducing activity, superiorly stable, readily hydrolyzablein vivo, and satisfactorily high in physiological activity, as well asdeveloping its preparation and uses in foods, beverages, pharmaceuticalsfor susceptive diseases, and cosmetics.

It was also found that since, when L-ascorbic acid is ingested with anα-glucosyl saccharide, 2-O-α-D-glucopyranosyl-L-ascorbic acid issynthesized and then metabolized in vivo, it would be an ideallyconvenient, novel saccharide derivative of L-ascorbic acid in view ofits safety.

A powder which is obtainable by concentrating and pulverizing an aqueoussolution of 2-O-α-D-glucopyranosyl-L-ascorbic acid is amorphous andstrongly hygroscopic, and has the drawback that it readily absorbsmoisture under ambient conditions to cause deliquescence andconsolidation.

SUMMARY OF THE INVENTION

Accordingly, another object of the present invention is to overcome thedrawback of such an amorphous 2-O-α-D-glucopyranosyl-L-ascorbic acid, inparticular, to provide a satisfactorily free flowing powder which isfree from substantial hygroscopicity and consolidation under ambientconditions.

We studied 2-O-α-D-glucopyranosyl-L-ascorbic acid solids which exhibit asubstantial nonhygroscopicity under ambient conditions which is enoughto overcome the drawback of amorphous 2-O-α-D-glucopyranosyl-L-ascorbicacid.

As the result, we discovered a novel substance, a crystalline2-O-α-D-glucopyranosyl-L-ascorbic acid, as well as finding that itprovides a substantially nonhygroscopic, satisfactorily free flowing,anhydrous crystalline powder which causes neither deliquescence norconsolidation under ambient conditions. Further, we developed a processto prepare such a crystalline 2-O-α-D-glucopyranosyl-L-ascorbic acid,and also processes to prepare foodstuffs, pharmaceuticals for susceptivediseases and cosmetics which all contain the same.

BRIEF EXPLANATION OF THE FIGURES

FIG. 1 is the uv-absorption spectrum of a crystalline2-O-α-D-glucopyranosyl-L-ascorbic acid according to the invention whenin a solution at pH 2.0.

FIG. 2 is the uv-absorption spectrum of the crystalline2-O-α-D-glucopyranosyl-L-ascorbic acid when in a solution at pH 7.0.

FIG. 3 is the infrared absorption spectrum of the crystalline2-O-α-D-glucopyranosyl-L-ascorbic acid.

FIG. 4 is the infrared absorption spectrum of an amorphous2-O-α-D-glucopyranosyl-L-ascorbic acid as the control.

FIG. 5 is the microscopic view of a crystal grown in a supersaturatedsolution (×40).

FIG. 6 is the ORTEP figure of a crystalline2-O-α-D-glucopyranosyl-L-ascorbic acid according to the invention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is feasible with any2-O-α-D-glucopyranosyl-L-ascorbic acid, regardless of its preparationprocess such as biochemical and organic chemical processes.

In view of safety and economical efficiency,2-O-α-D-glucopyranosyl-L-ascorbic acid is desirably formed by abiochemical process wherein a saccharide-transferring enzyme is allowedto act alone or together with glucoamylase on a solution containingL-ascorbic acid and an α-glucosyl saccharide.

The wording "exhibiting no direct reducing activity" means that unlikeL-ascorbic acid, a saccharide derivative thereof does not reduce anddecolor 2,6-dichlorophenolindophenol intact.

The wording "L-ascorbic acid" as referred to in the present inventionmeans L-ascorbates such as alkaline metal salts, alkaline earth metalsalts and mixtures thereof, and should not be restricted to freeL-ascorbic acid, as far as the present invention is feasible therewith.Thus, if necessary, such as sodium L-ascorbate and calcium L-ascorbateare suitably usable in the saccharide-transfer reaction, as well as freeL-ascorbic acid.

The wordings "α-glycosyl-L-ascorbic acid" and"2-O-α-D-glucopyranosyl-L-ascorbic acid" mean, in addition to those infree acid form, as far as the present invention is feasible therewith.

The α-glucosyl saccharides usable in the invention are those whichpermit a saccharide-transferring enzyme to form from L-ascorbic acid anα-glycosyl-L-ascorbic acid wherein equimolar or more α-D-glucosylresidues are bound to L-ascorbic acid. For example,maltooligosaccharides such as maltose, maltotriose, maltoteraose,maltopentaose, maltohexaose, maltoheptaose and maltooctaose are suitablychosen, as well as partial starch hydrolysates such as dextrin,cyclodextrin and amylose, liquefied starch, gelatinized starch, andsolubilized starch.

Consequently to facilitate the formation of α-glycosyl-L-ascorbic acid,one should choose an α-glucosyl saccharide which is susceptible to thesaccharide-transferring enzyme to be used.

For example, when α-glucosidase (EC 3.2.1.20) is used as thesaccharide-transferring enzyme, maltooligosaccharides such as maltose,maltotriose, maltotetraose, maltopentaose, maltohexaose, maltoheptaoseand maltooctaose are suitable, as well as partial starch hydrolysatesand dextrins with a DE (Dextrose Equivalent) of about 5-60. Whencyclomaltodextrin glucanotransferase (EC 2.4.1.19) is used as thesaccharide-transferring enzyme, partial starch hydrolysates such asgelatinized starches with a DE below 1 and dextrins with a DE up to 60are suitable. When α-amylase (EC 3.2.1.1) is used as thesaccharide-transferring enzyme, partial starch hydrolysates such asgelatinized starch with a DE below 1 and dextrins with a DE up to about30 are suitable.

The concentration of L-ascorbic acid during the reaction is generally 1w/v % or higher, preferably, about 2-30 w/v %, while the concentrationof an α-glucosyl saccharide is generally about 0.5- to 30-fold higherthan that of L-ascorbic acid.

The saccharide-transferring enzymes usable in the invention are thosewhich transfer one or several α-glucosyl groups at least to the numbertwo carbon atom in L-ascorbic acid without decomposing it when allowedto act on a solution which contains L-ascorbic acid and an α-glucosylsaccharide having an adequate susceptivity to the enzyme.

For example, α-glucosidases derived from animals, plants andmicroorganisms such as those from mouse kidney, rat intestinal mucosa,dog small intestine, pig small intestine, rice seed, maize seed, andthose from a culture which is obtainable by cultivating in a nutrientculture medium yeasts and bacteria of the genera Mucor, Penicillium andSaccharomyces; cyclomaltodextrin glucanotransferases from a culture ofbacteria such as those of the genera Bacillus and Klebsiella; andα-amylase from a culture of bacteria such as those of the genus Bacillusare suitably chosen.

Such a saccharide-transferring enzyme need not necessarily be purifiedprior to its use, as long as it fulfills the above requirements.Generally, the present invention is feasible with a crude enzyme. Ifnecessary, saccharide-transferring enzymes can be purified byconventional method, prior to its use. Of course, commercializedsaccharide-transferring enzymes can be used in the invention. The amountof a saccharide-transferring enzyme and reaction time are closelydependent upon each other. With an economical viewpoint,saccharide-transferring enzyme is used in an amount which completes thereaction within about 3-80 hours.

Immobilized saccharide-transferring enzymes are favorably usablebatchwise and in continuous manner.

The reaction process according to the invention is usually carried outby adding a saccharide-transferring enzyme to a solution containing theabove described L-ascorbic acid and an α-glucosyl saccharide, andkeeping the mixture under conditions where the enzyme is substantiallyactive; usually, at a pH in the range of about 3-9 and a temperature inthe range of about 20°-80° C. Since during the reaction, L-ascorbic acidtends to cause an oxidative decomposition, it is desirable to keep themixture under conditions which shield aeration and light as far aspossible so that L-ascorbic acid is in its reducing form. The reactionis favorably carried out in the presence of such as thiourea andhydrogen sulfide, if necessary.

The desired substance can be obtained by incorporating L-ascorbic acidand an α-glucosyl saccharide in the culture of a growing microorganismwhich is capable of producing a saccharide-transferring enzyme.

Generally, the reaction process according to the invention is carriedout by allowing a saccharide-transferring enzyme to act alone ortogether with glucoamylase on a solution which contains L-ascorbic acidand an α-glucosyl saccharide.

Such a process is feasible with glucoamylases derived from varioussources such as microorganisms and plants. Usually, commercializedglucoamylase derived from a microorganism of the genera Aspergillus andRhizopus are suitable.

To form 2-O-α-D-glucopyranosyl-L-ascorbic acid, glucoamylase can be usedsimultaneously with a saccharide-transferring enzyme. Generally toimprove the reaction efficiency, desirably, a saccharide-transferringenzyme is first used to transfer equimolar or more D-glucose residues toL-ascorbic acid to form an α-glycosyl-L-ascorbic acid, then glucoamylaseis used to accumulate 2-O-α-D-glucopyranosyl-L-ascorbic acid. β-Amylase(EC 3.2.1.2) can be freely used along with glucoamylase.

Alpha-glycosyl-L-ascorbic acids formed by such a saccharide-transferringenzyme bear an α-D-glucosyl group consisting of 1-7 glucosyl groupslinked via the α-1,4 fashion, and such an α-D-glucosyl group is bound atleast to the primary alcohol group which is located at the number twocarbon atom. Particular substances are, for example,2-O-α-D-glucosyl-L-ascorbic acid, 2-O-α-D-maltosyl-L-ascorbic acid,2-O-α-maltotriosyl-L-ascorbic acid, 2-O-α-D-maltotetraosyl-L-ascorbicacid, 2-O-α-D-maltopentaosyl-L-ascorbic acid,2-O-α-D-maltohexaosyl-L-ascorbic acid and2-O-α-D-maltoheptaosyl-L-ascorbic acid. Although α-glucosidase generallyforms only 2-O-α-D-glucosyl-L-ascorbic acid, 2-O-α-D-maltosyl-L-ascorbicacid and 2-O-α-D-maltotriosyl-L-ascorbic acid can be formed in mixture,if necessary.

In the case of using either cyclomaltodextrin glucanotransferase orα-amylase, α-glycosyl-L-ascorbic acids with a higher α-D-glucosyl groupare formed in mixture. Dependently on the α-glucosyl saccharide,cyclomaltodextrin glucanotransferase yields an α-D-glucosyl group with apolymerization degree distributing in the range of 1-7, while α-amylaseyields a slight narrower distribution. Such a mixture can be partiallyhydrolyzed with either of α-amylase (EC 3.2.1.1), β-amylase (EC 3.2.1.2)and glucoamylase (EC 3.2.1.3) to reduce the polymerization degree of theα-D-glucosyl group, if necessary. For example,2-O-α-D-maltosyl-L-ascorbic acid and higher polymers are hydrolyzed toaccumulate 2-O-α-D-glucosyl-L-ascorbic acid when subjected toglucoamylase. β-Amylase predominantly hydrolyzes2-O-α-D-maltotetraosyl-L-ascorbic acid and higher polymers to accumulate2-O-α-D-glucosyl-L-ascorbic acid, 2-O-α-D-maltosyl-L-ascorbic acid and2-O-α-D-maltotriosyl-L-ascorbic acid in mixture.

Reaction mixtures obtained by these methods usually contains theremaining L-ascorbic acid, D-glucose and α-glucosyl saccharide togetherwith 2-O-α-D-glucopyranosyl-L-ascorbic acid.

When needed as a refined product with a high2-O-α-D-glucopyranosyl-L-ascorbic acid content, such a reaction mixtureis subjected to one or more separations methods wherein the differencebetween 2-O-α-D-glucopyranosyl-L-ascorbic acid and contaminants such asremaining L-ascorbic acid, D-glucose and α-glucosyl saccharides inmolecular weight and/or affinity is utilized; for example, membraneseparation, gel filtration chromatography, column chromatography,high-performance liquid chromatography (HPLC) and ion exchangechromatography. In this case, the separated L-ascorbic acid andα-glucosyl saccharide can be favorably reused as a starting material inthe saccharide-transfer reaction. If necessary, after completion of thesaccharide-transfer reaction but before separation such as bychromatography, the reaction mixture can be treated by one or moremethods; for example, a method wherein the reaction mixture is heatedand the insolubilized substances are removed by filtration; anothermethod wherein the reaction mixture is treated, for example, withactivated carbon to adsorb the proteinaceous and coloring substances fortheir removal; and another method wherein the reaction mixture isdemineralized with cation exchange resin (H⁺ -form), and treated withanion exchange resin (OH⁻ -form) to remove anions and salts byadsorption.

The following will explain the crystallization of2-O-α-D-glucopyranosyl-L-ascorbic acid. Crystallizable2-O-α-D-glucopyranosyl-ascorbic acid is usually in the form of asupersaturated solution, and any 2-O-α-D-glucopyranosyl-L-ascorbic acidspecimen can be used regardless of its preparation process, as far as acrystalline 2-O-α-D-glucopyranosyl-L-ascorbic acid is obtainabletherefrom. The degree of supersaturation is usually set to about1.05-1.5. More particularly, 2-O-α-D-glucopyranosyl-L-ascorbic acid,purity of about 75% or higher, is prepared into an about 65-95 w/w %aqueous solution, and the temperature is set to a level which does notfreeze the solution and causes a less heat loss during the processing,desirably, in the range of 0°-95° C. The degree of supersaturation andviscosity are controllable by the addition of such as methanol, ethanoland acetone. A supersaturated solution of2-O-α-D-glucopyranosyl-L-ascorbic acid is placed at a relatively hightemperature such as 20°-60° C. in a crystallizer, added with a seedcrystal, desirably, in an amount of 0.1-10 w/w %, and crystallized intomassecuite while accelerating the crystallization by gentle stirring. Inthis way, the crystalline 2-O-α-D-glucopyranosyl-L-ascorbic acid of theinvention is easily obtainable by seeding a supersaturated solution of2-O-α-D-glucopyranosyl-L-ascorbic acid. To prepare the resultantmassecuite into final crystalline product, conventional methods, forexample, separation, block-pulverization, spray-drying and fluidized-bedgranulation methods are employable.

The separation method, wherein a massecuite is usually separated intocrystalline 2-O-α-D-glucopyranosyl-L-ascorbic acid and mother liquor(molasses) with a basket-type centrifuge, is convenient in thepreparation of a nonhygroscopic high-purity crystalline2-O-α-D-glucopyranosyl-L-ascorbic acid, and the separated crystal can bewashed by spraying thereto a small amount of water when a much higherpurity is needed. The separated mother liquor is purified andconcentrated similarly as above into a massecuite which is then reusedto recover such as second and third crystalline crops. This improves theyield of crystalline 2-O-α-D-glucopyranosyl-L-ascorbic acid.

Since the other three methods do not remove molasses, they never improvethe purity of crystalline 2-O-α-D-glucopyranosyl-L-ascorbic acid infinal powdery products, but realize a high product yield. Accordingly,such a product usually contains, for example, L-ascorbic acid,2-O-α-D-maltosyl-L-ascorbic acid, higher α-glycosyl-L-ascorbic acids,glucose and α-glucosyl saccharides along with crystalline2-O-α-D-glucopyranosyl-L-ascorbic acid.

In the case of spray-drying, usually, a massecuite with bothconcentration of 70-85 w/w % and crystallinity of 25-60 w/w % isspray-dried through a high-pressure nozzle, and the obtained dropletsare dried in an air stream of a temperature, for example, 30°-60° C.,which does not melt crystalline powder, and then aged in a stream of30°-60° C. air for about 1-20 hours. Thus, a nonhygroscopic or scarcelyhygroscopic crystalline powder is obtainable with an ease. In the caseof block-pulverization, usually, a massecuite with both moisture contentof 5-15 w/w % and crystallinity of about 10-60 w/w % is allowed to standfor 0.5-5 days so that the whole content is crystallized and solidifiedinto block. Cutting and drying of the resultant block readily yield anonhygroscopic or scarcely hygroscopic crystalline powder.

Alternatively, 2-O-α-D-glucopyranosyl-L-ascorbic acid in solution isconcentrated by heating into a supersaturated solution in melting form,moisture content below 5 w/w %, which is then kneaded along with a2-O-α-D-glucopyranosyl-L-ascorbic acid seed crystal at a temperaturebelow its melting point, and prepared into a desired form, for example,powder, granule, rod, plate and cube. Thus, a nonhygroscopic or scarcelyhygroscopic crystalline solid is obtainable. Dependent on both purityand crystallinity, the crystalline 2-O-α-D-glucopyranosyl-L-ascorbicacid obtained in this way is substantially nonhygroscopic or scarcelyhygroscopic, free flowing, and free of adhesion. Some of its advantagesare as listed below:

(1) It exhibits no direct reducing activity, and is extremely stable.Unlike L-ascorbic acid, it scarcely causes the Maillard reaction.Because of these characteristics, it effects no undesired reaction whenmixed with such as protein, lipid, saccharide and physiologically-activesubstance, but stabilizes these substances.

(2) It is susceptible to hydrolysis to form L-ascorbic acid, and thiselicits the same reductant and antioxidant activities as L-ascorbicacid.

(3) It is readily hydrolyzable by the in vivo enzyme system intoD-glucose and L-ascorbic acid, and thus the physiological activitiesinherent to L-ascorbic acid are elicited.

(4) It is highly safe because it is synthesized and then metabolized invivo when L-ascorbic acid is ingested together with an α-glucosylsaccharide.

(5) It is substantially nonhygroscopic or scarcely hygroscopic, butexhibits a high dissolution rate or solubility in water. Because ofthese characteristics, it is favorably usable as a vitamin C-enrichingagent, taste-improving agent, souring agent and stabilizer in foods andbeverages, in powder, granule and tablet, such as vitamin compound,cream filling, chocolate, chewing gum, instant juice and seasoning mix.

(6) It is substantially nonhygroscopic or scarcely hygroscopic, freeflowing, and free from consolidation. Thus, it is much more easilyhandled than amorphous product, and this considerably cuts the materialand labor costs in packaging, transportion and storage.

Because of advantages, crystalline 2-O-α-D-glucopyranosyl-L-ascorbicacid can be favorably incorporated as a stabilizer, taste-improvingagent, souring agent, antioxidant, quality-improving agent,uv-absorbent, and preventive and remedy for susceptive diseasesincluding viral diseases, bacterial diseases, circulatory diseases andmalignant tumors, desirably in an amount of 0.001 w/w % or more, alongor in combination with one or more ingredients in foods, beverages,feeds, pet foods, pharmaceuticals for susceptive diseases, and cosmeticssuch as skin-refining agents and skin-whitening agents, as well as inagents directed to enrich a highly-safe, natural vitamin C. In thiscase, L-ascorbic acid, vitamin E, rutin, α-glycosyl rutin and/orhesperidin are favorably usable in combination with crystalline2-O-α-D-glucopyranosyl-L-ascorbic acid.

Since crystalline 2-O-α-D-glucopyranosyl-L-ascorbic acid is highlyresistant to acid and heat, and well harmonizes with various substanceswhich taste sour, salty, bitter, delicious and astringent, it isfavorably usable as a vitamin C-enriching agent, taste-improving agent,antioxidant and quality-improving agent in foods and beverages ingeneral, for example, seasonings such as soy sauce, say sauce powder,miso, miso powder, "moromi", "hishio", "furikake", mayonnaise, dressing,vinegar, "sanbai-zu", "funmatsu-sushi-su", "chuka-no-moto", "tentsuyu(soup for tenpura)", "mentsuyu (soup for Japanese-style noodles)",Worcester sauce, ketchup, "yakiniku-no-tare (soup for grilled meat)",curry roux, stew premix, soup premix, "dashi-no-moto", mixed seasoning,"mirin (heavily sweetened sake)", "shin-mirin (synthetic mirin)", tablesugar and coffee sugar; Japanese-style confectioneries such as "senbei(rice crackers)", "arare (pellet-shaped senbei)", "okoshi (millet-andrice cracker)", "karinto (fried dough cookie)", "gyuhi (starch paste)",rice paste, "manju (bun with a bean-jam filling)", "uiro (sweet ricejelly)", "an (bean jam)", "yokan (sweet jelly of beans)", "mizu-yokan(soft adzuki-bean jelly)", " kingyoku", jelly, castella and "amedama(Japanese-style toffee)"; Western-style confectioneries such as bun,biscuit, cracker, cookie, pie, pudding, cream puff, waffle, sponge cake,doughnut, chocolate, chewing gum, caramel and candy; frozen dessertssuch as ice cream and sherbet; syrups such as those for fruit preserveand "kaki-gori (shaved ice)"; spreads and pastes such as butter cream,custard cream, flour paste and fruit paste; processed fruits such asjam, marmalade, syrup-preserved fruit and crystallized fruit; processedfoods such as those of fruits and vegetables; cereals such as bakeryproduct, noodle, vermicelli, boiled rice and synthetic meat; fatty foodsubstances such as salad oil and margarine; pickled products such as"fukujin-zuke (sliced vegetables picked in soy sauce)", "bettara-zuke(fresh radish pickles)", "senmai-zuke" and "rakkyo-zuke (pickledshallots)"; premixes for pickled products such as "takuan-zuke-no-moto"and "hakusai-zuke-no-moto"; meat products such as ham and sausage; fishmeat products such as fish meat ham, fish meant sausage, "kamaboko(boiled fish paste)", "chikuwa (literally bamboo wheels)" and "hanpen";relishes such as "uni-no-shiokara (salted guts of sea urchin)","ika-no-shiokara (salted guts of squid)", "su-konbu", "saki-surume" and"fugu-no-mirinboshi"; "tsukudani (food boiled down in soy sauce)" suchas those of "nori (dried seaweed)", "sansai (mountain vegetables)","surume (dried squid)", small fish and shellfish; daily dishes such as"nimame (cooked beans)", potato salad, "konbu-maki (tangle roll)" and"tenpura (deep-fried foods)"; egg and milk products such as"kinshi-tamago", milk beverage, butter and cheese; bottled and cannedproducts such as those of meat, fish meat, fruit and vegetable;alcoholic drinks such as synthetic sake, "zojo-shu", liqueur, wine andwhisky; beverages such as coffee, cocoa, juice, carbonated beverage,lactic acid beverage and lactobacillus beverage; and premixes andinstant foodstuffs such as pudding premix, hot cake premix, instantjuice, instant coffee, "sokuseki-shiruko (premix of adzuki-bean soupwith rice cake)" and instant soup. Furthermore, crystalline2-O-α-D-glucopyranosyl-L-ascorbic acid can be favorably incorporated infeeds and pet foods for domestic animals and poultries including honeybee, silkworm and pet fish for the enrichment of vitamin C, theimprovement of their taste qualities and the prevention of oxidation.

Also 2-O-α-D-glucopyranosyl-L-ascorbic acid can be favorablyincorporated in special foods and beverages, preventives and remediesfor susceptive diseases, cosmetics including skin-refining agent andskin-whitening agent, for example, cigar, cigarette, troche, cod-liveroil drop, vitamin compound, oral refreshing agent, cachou, gargle,intubation nutrient, internal medicine, injection, dentifrice, lipstick,eye shadow, milky lotion, moisture liquid, cosmetic cream, foundation,sunscreen agent, cleansing soap, shampoo and rinse, in addition to theuses as uv-absorbent and deterioration-preventing agent for plastics andalso as a substrate for assaying glycoside hydrolases.

The wording "susceptive diseases" as referred to in the invention meansthose which are prevented and/or treated with crystalline2-O-α-D-glucopyranosyl-L-ascorbic acid and its solution; for example,viral diseases, bacterial diseases, traumatic diseases, immunopathies,allergy, diabetes, cataract, circulatory diseases and malignant tumors.The shape and form of pharmaceuticals for susceptive diseases can befreely chosen to meet to their final use; for example, liquidpharmaceuticals such as nebula, collyrium, collunarium, collutory andinjection, paste pharmaceuticals such as ointment, cataplasm and cream,and solid pharmaceuticals such as powder, granule, capsule and tablet.In the preparation of such a pharmaceutical, one or more ingredients,for example, remedy, biologically-active substance, antibiotic,adjuvant, filler, stabilizer, coloring agent and flavoring agent, can besuitably used in combination, if necessary.

The dose is adequately changed dependently on the2-O-α-D-glucopyranosyl-L-ascorbic acid content, administration route andadministration frequency; usually, in the range of about 0.001-100g/day/adult as 2-O-α-D-glucopyranosyl-L-ascorbic acid.

Cosmetics can be prepared similarly as in pharmaceuticals.

Crystalline 2-O-α-D-glucopyranosyl-L-ascorbic acid is incorporated inproducts by conventional method, for example, mixing, kneading,dissolving, melting, soaking, permeating, spreading, applying, coating,spraying, injecting, crystallizing and solidifying, before completion oftheir processing.

Crystalline 2-O-α-D-glucopyranosyl-L-ascorbic acid is favorably usableas a material for chemical reactions which are effected under anhydrousconditions because it is substantially anhydrous, and its completeanhydrousness is attainable by a brief ventilation of hot air. Thus, bysubjecting a crystalline 2-O-α-D-glucopyranosyl-L-ascorbic acid toconventional chemical reaction under anhydrous conditions, for example,its ether and ester derivatives can be prepared with an ease. Thesederivatives are favorably usable, for example, in surface active agent,emulsifier, stabilizer and lipophilic vitamin C.

When a crystalline 2-O-α-D-glucopyranosyl-L-ascorbic acid is in freeacid form, it can be, if necessary, converted, for example, into sodiumsalt, calcium salt, magnesium salt, iron salt, copper salt and zinc saltby allowing it to react with an aqueous solution of such as metalhydroxide and metal carbonate, so that the resultant substance isimparted with abilities of adequately adjusting pH and also exhibitingthe activities of minerals and vitamin C. Such a substance is favorablyusable in nutritive fortifiers and chemical agents.

The following experiments will explain in detail a typical crystalline2-O-α-D-glucopyranosyl-L-ascorbic acid according to the invention.

EXPERIMENT 1 Preparation of Crystalline2-O-α-D-Glucopyranosyl-L-Ascorbic Acid

Nine parts by weight of dextrin (DE about 6) was dissolved in 15 partsby weight of water by heating, and the solution was added with 3 partsby weight of L-ascorbic acid under reducing conditions, further addedwith 400 units/g dextrin of cyclomaltodextrin glucanotransferasecommercialized by Hayashibara Biochemical Laboratories, Inc., Okayama,Japan, and allowed to react for 24 hours while keeping the solution atpH 5.5 and 60° C. The reaction mixture was fed to "AQ-303 ODS" HPLCsystem, a product of Yamamura Chemical Laboratories Co., Ltd., Kyoto,Japan, equipped with "LC-6" column, a product of Shimadzu SeisakushoLtd., Kyoto, Japan, and eluted with 0.1M KH₂ PO₄ -H₃ PO₄ buffer (pH 2.0)at a flow rate of 0.5 ml/minute while monitoring with "MULT-340"detector system, a product of Japan Spectroscopic Co., Ltd., Tokyo,Japan. As the result, L-ascorbic acid appeared at a retension time of9.5 minutes, while the newly formed α-D-glucosyl-L-ascorbic acid,α-D-maltosyl-L-ascorbic acid, α -D-maltotriosyl-L-ascorbic acid,α-D-maltotetraosyl-L-ascorbic acid, α-D-maltopentaosyl-L-ascorbic acid,α-D-maltohexaosyl-L-ascorbic acid and α-D-maltoheptaosyl-L-ascorbic acidappeared at respective retension time of 11.2 minutes, 15.7 minutes,20.6 minutes, 24.9 minutes, 28.1 minutes, 32.1 minutes and 38.6 minutes.About 60% of the L-ascorbic acid was converted intoα-glycosyl-L-ascorbic acid. Thereafter, the reaction mixture wasfiltered with UF membrane to remove the enzyme, adjusted to pH 5.0 and55° C., added with 10 units/g dextrin of glucoamylase (EC 3.2.1.3)commercialized by Seikagaku Kogyo Ltd., Tokyo, Japan, and allowed toreact for 24 hours. HPLC analysis of the reaction mixture revealed thatα-D-maltosyl-L-ascorbic acid and higher α-glycosyl-L-ascorbic acids werehydrolyzed into 2-O-α-D-glucopyranosyl-L-ascorbic acid.

The reaction mixture was then heated to inactive the remaining enzyme,decolored and filtered with activated carbon, and the filtrate wasconcentrated to about 50 w/w %.

The concentrate was subjected to column chromatographed on "XT-1016 (Na⁺-form)", a strongly-acidic cation exchange resin commercialized by TokyoChemical Industries, Tokyo, Japan, in accordance with the methoddisclosed in Japanese Patent Laid-Open No. 23,799/83 with a slightmodification to elute and recover a 2-O-α-D-glucopyranosyl-L-ascorbicacid-rich fraction, purity of about 94%, which was then purified by thedemineralization using a cation exchange resin (H⁺ -form), concentratedto about 80 w/w %, placed in a glass vessel, and allowed to stand at20°-35° C. for about 1 month. Thus, crystallization occurred. A portionof the crystal was added to a fresh preparation of the same purified andconcentrated 2-O-α-D-glucopyranosyl-L-ascorbic acid-rich fraction, andcrystallized by gentle stirring. The resultant massecuite was separatedinto the molasses and crystal, and the latter was then washed byspraying thereto a small amount of chilled water for a higher purity,dissolved in water and recrystallized. Thus, a high-purity crystal,purity of about 99.9% or higher, was obtained.

EXPERIMENT 2 Physiochemical Properties of Crystalline2-O-α-D-Glucopyranosyl-L-Ascorbic Acid

Characterization of a crystal, obtained by the recrystallization inaccordance with the method in Experiment 1, revealed that it was a novelanhydrous crystalline 2-O-α-D-glucopyranosyl-L-ascorbic acid.

The properties of the crystal will be described hereinafter.

(1) Elemental analysis: Found; C=42.6%, H=5.36%, Calculated; C=42.4%,H=5.37%, N<0.01%, (for chemical formula C₁₂ H₁₈ O₁₁).

(2) Molecular weight; FD mass spectrometric analysis with "M-80B", amass spectrometry commercialized by Hitachi Ltd., Tokyo, Japan, revealeda (M+H)⁺ peak at 339 (molecular weight for chemical formula C₁₂ H₁₈ O₁₁is 338).

(3) Melting point: 158.5°-159.5° C.

(4) Heat of dissolution: Endothermic (27.2 kcal/g).

(5) Specific rotation: [α]_(D) ²⁰ =+189.6° (H₂ O, pH 1.98); [α]_(D) ²⁰=+246.3° (H₂ O, pH 7.10).

(6) uv-Absorption spectrum: uv-absorption spectrum was determined in 50μM solution. The spectrum at pH 2.0 was as shown in FIG. 1, while thatat pH 7.0 was as shown in FIG. 2.

    λ(max)=238 nm, ε=0.93×10.sup.4 (pH 2.0)

    λ(max)=260 nm, ε=1.50×10.sup.4 (pH 7.0)

(7) Infrared absorption spectrum: The KBr tablet method was used. Theinfrared spectrum of the crystal was as shown in FIG. 3; while that ofan amorphous substance as the control was as shown in FIG. 4.

(8) Solubility: One hundred and twenty-five grams of the crystaldissolves in 100 g water at 25° C.

(9) Solubility in solvents: Readily soluble in water, 0.1N sodiumhydroxide and 0.1N acetic acid; soluble in methanol and ethanol; andinsoluble in ether, benzene, chloroform and ethyl acetate.

(10) Dissociation constant The pKa is 3.0. Comparison of this to thosefor various derivatives of L-ascorbic acid in Table 1 in J. Jernow etal., Tetrahedron, Vol. 35, pp. 1,483-1,486 (1979) and in Table 2 inPao-Wen Lu et al., Journal of Agricultural Food Chemistry, Vol. 32, pp.21-28 (1984) suggests that in the substance of the invention, thealcohol group which is located at the number two carbon atom in theascorbic acid moiety is responsible for the α-D-glucosyl linkage, whilethe alcohol group which is located at the number three carbon atom is infree form.

(11) Methylation analysis: The crystal was methylated by the methoddescribed in Pao-Wen Lu et al., Journal of Agricultural Food andChemistry, Vol. 32, pp. 21-28 (1984) wherein L-ascorbic acid wasmethylated with diazomethane to predominantly form 3-O-methyl-L-ascorbicacid. A subsequent hydrolysis of the resultant led to the formation of3-O-methyl-L-ascorbic acid and D-glucose as the predominant products.

(12) Physical properties and color: Colorless and transparent crystal.When pulverized, the crystal exhibits a sour taste, but exhibits noodor. Free of hygroscopicity and deliquescence. Loss on drying at 130°C. for 2 hours is less than 0.5 w/w %. FIG. 5 is the microscopic view ofa crystal growing in a supersaturated solution.

(13) Coloring reaction: Exhibiting no direct reducing activity, and notreducing and decoloring 2,6-dichlorophenolindophenol. Negative to the2,4-dinitrophenylhydrazine reaction. Turning green on theanthrone-sulfuric acid reaction.

(14) Structual element: Hydrolyzable by α-glucosidase or by treatmentwith 1N hydrochloric acid at 100° C. for 5 minutes to form L-ascorbicacid and D-glucose at a molar ratio of 1:1.

(15) Powder x-ray diffraction analysis: On powder x-ray diffractionanalysis using "GEIGERFLEX RAD-II B (CuKα ray)", a product of RigakuCorp., Tokyo, Japan, the crystal exhibits predominant diffraction angles(2θ) of 10.3°, 14.8°, 16.2°, 18.4° and 24.5°.

(16) x-Ray analysis on single crystal: x-Ray diffraction analysis on asingle crystal grown in a supersaturated solution revealed that it wasgrouped into the orthorhombic system, and its space group was P2₁ 2₁ 2₂with lattice constants of a=11.929 Å, b=24.351 Å and c=4.864 Å(α=β=γ=90°). These data evidently show that in the chemical structure ofthe crystal, an α-glucosyl linkage is formed between the alcohol groupat the number two carbon atom and the alcohol group which is located atthe number one carbon atom in D-glucose. FIG. 6 is the ORTEP figure ofthe crystal.

The above data suggest that the crystal is a novel crystalline2-O-α-D-glucopyranosyl-L-ascorbic acid shown by the formula [III]:##STR3##

Consequently, it is suggested that α-glycosyl-L-ascorbic acid whereinequimolar or more D-glucose residues are bound to L-ascorbic acid hasthe chemical structure shown by the formula [IV]: ##STR4## wherein n isan integer from 0 to 6.

EXPERIMENT 3 Stability of Crystalline 2-O-α-D-Glucopyranosyl-L-AscorbicAcid in Solution

2-O-α-D-Glucosyl-L-ascorbic acid was compared respectively to the6-O-α-D-glucosyl-L-ascorbic acid disclosed in Japanese PatentPublication No. 38,158/73, and L-ascorbic acid for their stability inaqueous solution. More particularly, each sample was adjusted to aconcentration of 70 micromoles and to pH 7.0 or 2.0, placed in acuvette, and measured for its absorbance at either 260 nm and pH 7.0 orat 245 nm and pH 2.0 while keeping the solution at 45° C. The remainingratio (%) was calculated with the absorbance.

The results were as shown in Table I.

As obvious from the results in Table I, unlike6-O-α-D-glucosyl-L-ascorbic acid and L-ascorbic acid, crystalline2-O-α-D-glucopyranosyl-L-ascorbic acid is extremely stable even inaqueous solution.

                  TABLE I                                                         ______________________________________                                               Time (hour)                                                            pH       0       1       2     4     18    24                                 ______________________________________                                        7   2GAsA    100%    100%  100%  100%  100%  100%                                 6GAsA    100%    88%   72%   43%    7%   4%                                   AsA      100%    80%   61%   32%    1%   1%                               2   2GAsA    100%    100%  100%  100%  100%  100%                                 6GAsA    100%    99%   98%   90%   25%   12%                                  AsA      100%    98%   95%   88%   12%   4%                               ______________________________________                                         Note: 2GAsA is the symbol for the crystalline                                 2O-D-glucopyranosyl-L-ascorbic acid of the invention; 6GAsA, for              6O-D-glucosyl-L-ascorbic acid as a control; and AsA, for Lascorbic acid a     another control.                                                         

EXPERIMENT 4 Acute Toxicity

A crystalline 2-O-α-D-glucopyranosyl-L-ascorbic acid specimen, preparedby the method in Experiment 1, was orally administered to 7 week-old ddmice for acute toxicity test. As the result, no mouse died whenadministered with up to 5 g of the specimen, and higher dose wasdifficult.

These confirmed that the specimen was extremely low in toxicity.

The following Examples A and Examples B will illustrate the crystalline2-O-α-D-glucopyranosyl-L-ascorbic acid and its uses respectively.

EXAMPLE A-1 Crystalline 2-O-α-D-Glucopyranosyl-L-Ascorbic Acid

Nine parts by weight of α-cyclodextrin was dissolved in 20 parts byweight of water by heating, and the solution was added with 3 parts byweight of L-ascorbic acid under reducing conditions, thereafter whilekeeping the solution at pH 5.5 and 65° C., added with 100 units/gα-cyclodextrin of cyclomaltodextrin glucanotransferase commercialized byHayashibara Biochemical Laboratories, Inc., Okayama, Japan, and allowedto react for 40 hours. HPLC analysis of the reaction mixture revealedthat about 50% of the L-ascorbic acid was converted intoα-glycosyl-L-ascorbic acid similarly as in Experiment 1.

Thereafter, the reaction mixture was heated to inactivate the remainingenzyme, adjusted to pH 4.5 and 55° C., added with 50 units/gα-cyclodextrin of glucoamylase, and allowed to react for 24 hours. HPLCanalysis of the newly formed reaction mixture revealed thatα-maltosyl-L-ascorbic acid and higher α-glycosyl-L-ascorbic acids werehydrolyzed into 2-O-α-D-glucopyranosyl-L-ascorbic acid.

The reaction mixture was then heated to inactivate the remaining enzyme,decolored and filtered with activated carbon, and the filtrate wasapplied to a column of a cation exchange resin (H⁺ -form) fordemineralization, further applied to a column of an anion exchange resin(OH⁻ -form) to adsorb anions. Thereafter, the column was washed withwater and applied with 0.5N hydrochloric acid for elution, and theeluate was subjected to gel filtration chromatography on "HW-40", a gelproduct of Tosoh Corp., Tokyo, Japan, to recover a2-O-α-D-glucopyranosyl-L-ascorbic acid-rich fraction which was thenconcentrated in vacuo to about 73 w/w %, placed in a crystallizer, addedwith 1 w/w % 2-O-α-D-glucopyranosyl-L-ascorbic acid seed crystal,adjusted to 40° C., gradually cooled to 25° C. over a period of 2 dayswhile accelerating the crystallization by gentle stirring, and fed to abasket-type centrifuge to remove the molasses. The remaining crystal waswashed by spraying thereto a small amount of an aqueous ethanol toobtain a crystalline 2-O-α-D-glucopyranosyl-L-ascorbic acid, purity ofabout 99%, in the yield of about 35 w/w % against the startingL-ascorbic acid.

Although the product was slightly different in such as melting point andspecific rotation from a crystal obtained by the method in Experiment 1,the other properties were substantially the same.

The product is substantially nonhygroscopic, easily heandleable, free ofdirect reducing activity, and satisfactorily high in stability andphysiological activities. Thus, the product is favorably usable as ataste-improving agent, souring agent, stabilizer, quality-improvingagent, antioxidant, physiologically active agent, uv-absorbent,pharmaceutical material and chemical in foods, beverages,pharmaceuticals for susceptive diseases, cosmetics and chemicalreagents, as well as in agents directed to enrich vitamin C.

EXPERIMENT A-2 Crystalline 2-O-α-D-Glucopyranosyl-L-Ascorbic Acid

Thirty parts by weight of dextrin (DE about 6) was dissolved in 40 partsby weight of water by heating, and the solution was added with 7 partsby weight of L-ascorbic acid under reducing conditions, thereafter whilekeeping the solution at pH 5.6 and 60° C., added with 250 units/gdextrin of cyclodextrin glucanotransferase and allowed to react for 40hours. After analyzing the reaction mixture by HPLC similarly as inExample A-1, about 65% of the L-ascorbic acid was converted intoα-glycosyl-L-ascorbic acid similarly as in Experiment 1.

Thereafter, the reaction mixture was filtered with UF membrane to removethe enzyme, adjusted to pH 5.0 and 50° C., added with 100 units/gdextrin of glucoamylase, and allowed to react for 6 hours. HPLC analysisof the reaction mixture revealed that α-D-maltosyl-L-ascorbic acid andhigher α-glycosyl-L-ascorbic acids were converted into2-O-α-D-glucopyranosyl-L-ascorbic acid.

Thereafter, the reaction mixture was heated to inactivate the remainingenzyme and filtered, after which the filtrate was concentrated andchromatographed on a column of "DOWEX 50WX4 (Ca⁺⁺ -form)", astrongly-acidic cation exchange resin commercialized by Dow ChemicalCo., Midland, Mich., U.S.A., in accordance with the method in Experiment1 with a slight modification. The eluted2-O-α-D-glucopyranosyl-L-ascorbic acid-rich fraction was purified by thedemineralization with a cation exchange resin (H⁺ -form), concentratedin vacuo to about 77 w/w %, placed in a crystallizer, added with 2 w/w %seed crystal, adjusted to 45° C., and gradually cooled to 28° C. over aperiod of 2 days while accelerating the crystallization by gentlestirring. The resultant massecuite was separated similarly as in Example1 to obtain a crystalline 2-O-α-D-glucopyranosyl-L-ascorbic acid, purityof about 98%, in the yield of about 45 w/w % against the startingL-ascorbic acid.

Similarly as the product in Example A-1, the product is substantiallynonhygroscopic, easily handleable, free of direct reducing activity, andsatisfactorily high in stability and physiological activities. Thus, theproduct is favorably usable as a taste-improving agent, souring agent,moisture-retaining agent, stabilizer, quality-improving agent,anti-oxidant, biologically active agent, uv-absorbent, pharmaceuticalmaterial and chemical in foods, beverages, pharmaceuticals forsusceptive diseases and cosmetics, as well as in agents directed toenrich vitamin C.

EXAMPLE A-3 Crystalline 2-O-α-D-Glucopyranosyl-L-Ascorbic Acid

Similarly as in Example A-2, cyclomaltodextrin glucanotransferase andglucoamylase were allowed to react to obtain a reaction mixturecontaining 2-O-α-D-glucopyranosyl-L-ascorbic acid which was then heatedto inactivate the enzymes, decolored and filtered with activated carbon.The resultant filtrate was demineralized with a cation exchange resin(H⁺ -form) and chromatographed on a column of a strongly-acidic cationexchange resin (H⁺ -form) in accordance with the method in Experiment 1with a slight modification, followed by elution. The2-O-α-D-glucopyranosyl-L-ascorbic acid-rich fraction was recovered,concentrated to about 90 w/w %, placed in a crystallizer, added withabout 2 w/w % seed crystal, gently stirred for 30 minutes, transferredin a tray, and crystallized and solidified by 3-day standing at 25° C.The content was then removed from the tray, fed to a cutting pulverizer,and dried to obtain a crystalline 2-O-α-D-glucopyranosyl-L-ascorbicacid, purity of about 95%, in the yield of about 70 w/w % against thestarting L-ascorbic acid.

Similarly as the product in Example A-1, the product is substantiallynonhygroscopic, easily handled, free from direct reducing activity, andsatisfactorily high in stability and physiological activities. Thus, theproduct is favorably usable as a taste-improving agent, souring agent,stabilizer, quality-improving agent, antioxidant, physiologically activeagent, uv-absorbent, pharmaceutical material and chemical in foods,beverages, pharmaceuticals for susceptive disease, cosmetics andchemical reagents, as well as in agents directed to enrich vitamin C.

EXAMPLE A-4 Crystalline 2-O-α-D-Glucopyranosyl-L-Ascorbic Acid

A 2-O-α-D-glucopyranosyl-L-ascorbic acid-rich fraction, prepared by themethod in Example A-3, was concentrated to about 80 w/w %, placed in acrystallizer, added with about 2 w/w % seed crystal, and graduallycooled from 50° C. while accelerating the crystallization by gentlestirring. The resultant massecuite, crystallinity of about 35%, wassprayed through a nozzle, diameter of 1.5 mm, equipped at the top of aspraying tower with a high-pressure pump, pressure of 150 kg/cm².

Simultaneously, 85° C. air was passed from the top of the tower towardsa net conveyer, provided at the bottom of the tower, to collect thepulverized product on the net conveyer and also to gradually carry theresultant crystalline powder out of the tower over a period of about 30minutes while passing a stream of 40° C. air upwards through the net.

The crystalline powder was then placed in an ageing tower and aged for10 hours for crystallization and dehydration. Thus, a crystalline2-O-α-D-glucopyranosyl-L-ascorbic acid, purity of about 95%, wasobtained in the yield of about 70 w/w % against the starting L-ascorbicacid.

Similarly as the product in Example A-1, the product is substantiallynonhygroscopic, easily handleable, free of direct reducing activity, andsatisfactorily high in stability and physiological activities. Thus, theproduct is favorably usable as a taste-improving agent, souring agent,stabilizer, quality-improving agent, antioxidant, physiologically activeagent, uv-absorbent, pharmaceutical material and chemical in foods,beverages, pharmaceuticals for susceptive diseases and cosmetics, aswell as in agents directed to enrich vitamin C.

EXAMPLE A-5 Crystalline 2-O-α-D-Glucopyranosyl-L-Ascorbic Acid EXAMPLEA-5(1) Preparation of α-Glucosidase

Mucor javanicus IFO 4570 was inoculated and cultivated at 30° C. for 44hours under aeration-agitation conditions in 500 parts by weight of aliquid culture medium which contained water together with 4.0 w/v %maltose, 0.1 w/v % potassium phosphate monobasic, 0.1 w/v % ammoniumnitrate, 0.05 w/v % magnesium sulfate, 0.05 w/v % potassium chloride,0.2 w/v % polypeptone and 1 w/v % calcium carbonate which had beensterilized by heating and sterilely added to the water immediatelybefore the inoculation. After completion of the culture, the mycelia wasrecovered and immobilized in usual manner.

EXAMPLE A-5(2) Preparation of Crystalline2-O-α-D-Glucopyranosyl-L-Ascorbic Acid

Forty parts by weight of "SUNMALT®" a crystalline maltose commercializedby Hayashibara Co., Ltd., Okayama, Japan, was dissolved in 70 parts byweight of water by heating, and the solution was added with 10 parts byweight of L-ascorbic acid under reducing conditions, further added with10 units/g maltose of an immobilized α-glucosidase prepared by themethod in Example A-5(1), and allowed to react at pH 5.5 and 50° C. for3 hours under light-shielding conditions.

One unit of α-glucosidase is defined as the amount of enzyme thatreleases 1 micromole glucose at 37° C. over a time period of 1 minutewhen assayed under the following conditions. After appropriatelydiluting, 100 microliters of an enzyme solution is added to a mixturesolution of 250 microliters of 4 w/v % maltose and 750 microliters of0.1M acetate buffer (pH 6.0) containing 1.35 mM EDTA, and the mixture isallowed to react at 37° C. for 30 minutes, incubated in boiling waterfor 3 minutes to suspend the reaction, and centrifuged. Thereafter, 20microliters of the supernatant is sampled, added with 1 ml of "GLUCOSE BTEST", a coloring reagent for the glucose oxidase method commercializedby Wako Pure Chemical Industries, Ltd., Osaka, Japan, incubated at 37°C. for 20 minutes for color development, and assayed for absorbance at505 nm.

Thereafter, the reaction mixture was filtered to recover the immobilizedα-glucosidase which was reused in another reaction batch. The filtratewas decolored with activated carbon, and chromatographed on a column ofa strongly-acidic cation exchange resin by the method in Example A-2 torecover a 2-O-α-D-glucopyranosyl-L-ascorbic acid-rich fraction which wasthen purified with a cation exchange resin, thereafter in accordancewith the method in Example A-3, concentrated in vacuo to about 90 w/w %,placed in a crystallizer, added with a seed crystal, crystallized andsolidified in a tray, fed to a cutting pulverizer, and dried to obtain acrystalline 2-O-α-D-glucopyranosyl-L-ascorbic acid, purity of about 88%,in the yield of about 20 w/w % against the starting L-ascorbic acid.

Comparing to the product in Example A-1, the product is slightlyinferior, but substantially nonhygroscopic, easily handleable, free ofdirect reducing activity, and satisfactorily high in stability andphysiological activites. Thus, the product is favorably usable as ataste-improving agent, souring agent, stabilizer, quality-improvingagent, antioxidant, physiologically active agent, uv-absorbent,pharmaceutical material and chemical in foods, beverages,pharmaceuticals for susceptive diseases and cosmetics, as well as inagents directed to enrich vitamin C.

EXAMPLE B-1 Chewing Gum

Twenty-five parts by weight of gum base and 20 parts by weight of acrystalline 2-O-α-D-glucopyranosyl-L-ascorbic acid obtained by themethod in Example A-2 were kneaded at 60° C. with a mixer, and themixture was added with 50 parts by weight of "MABIT®", an anhydrouscrystalline maltitol commercialized by Hayashibara Shoji Inc., Okayama,Japan, 1.5 parts by weight of calcium phosphate and 0.1 part by weightof an L-menthol including β-cyclodextrin, and further mixed with a smallamount of seasoning, rolled and cut to obtain the captioned product. Theproduct is a vitamin C-enriched, low-cariogenic and low-caloric chewinggum.

EXAMPLE B-2 "Gyuhi (Starch Paste)"

One part by weight of waxy rice starch was mixed with 1.2 parts byweight of water, and the mixture was mixed to homogeneity with 1.5 partsby weight of sucrose, 0.7 parts by weight of "SUNMALT®", a crystallineβ-maltose commercialized by Hayashibara Co., Ltd., Okayama, Japan, 0.1part by weight of a crystalline 2-O-α-D-glucopyranosyl-L-ascorbic acidobtained by the method in Example A-5 while gelatinizing by heating.Thereafter, the resultant was molded and packaged in usual manner toobtain "gyuhi".

The product is a vitamin C-enriched, Japanese-style confectionery withexcellent flavor and biting properties, which looks like "kibi-dango(millet dumpling)". The product exhibits a long shelf life because itsretrogradation is effectively suppressed.

EXAMPLE B-3 Mixed Sweetener

A mixed sweetener was obtained by mixing 100 parts by weight of honey,50 parts by weight of isomerized sugar, 2 parts by weight of "kurozato(unrefined sugar)" and 1 part by weight of a crystalline2-O-α-D-glucopyranosyl-L-ascorbic acid obtained by the method in ExampleA-3.

The product is a vitamin C-enriched sweetener, and suitable for healthfood.

EXAMPLE B-4 Chocolate

Forty parts by weight of cacao paste, 10 parts by weight of cacaobutter, 50 parts by weight of anhydrous crystalline maltitol and 1 partby weight of a crystalline 2-O-α-D-glucopyranosyl-L-ascorbic acidobtained by the method in Example A-2 were mixed to homogeneity, and themixture was fed to a refiner to reduce the particle size, transferred toa conche, and kneaded therein at 50° C. for 2 days. In the kneadingstep, 0.5 parts by weight of lecithin was added and dispersed tohomogeneity. Thereafter, the content was adjusted to 31° C. with athermoregulator, and placed in a mold immediately before thesolidification of the butter, dearated with a vibrator, and solidifiedby passing it through a 10° C. cooling tunnel over a period of 20minutes. The content was removed from the mold, and packaged to obtainthe captioned product.

The product is free of hygroscopicity and excellent in color, gloss andtexture, as well as smoothly melting in the mouth to exhibit a moderateand mild sweetness and flavor. The product is a vitamin C-enriched,low-cariogenic and low-caloric chocolate.

EXAMPLE B-5 Cream Filling

A cream filling was obtained by mixing in usual manner 1,200 parts byweight of "FINETOSE®", a crystalline α-maltose commercialized byHayashibara Co., Ltd., Okayama, Japan, 1,000 parts by weight ofshortening, 10 parts by weight of a crystalline2-O-α-D-glucopyranosyl-L-ascorbic acid obtained by the method in ExampleA-4, 1 part by weight of lecithin, 1 part by weight of lemon oil and 1part by weight of vanilla oil to homogeneity.

The product is a vitamin C-enriched cream filling which is excellent intaste, flavor, melting and biting properties.

EXAMPLE B-6 Tablet

Twenty parts by weight of a crystalline2-O-α-D-glucopyranosyl-L-ascorbic acid obtained by the method in ExampleA-1 was mixed to homogeneity with 13 parts by weight of crystallineβ-maltose, 4 parts by weight of cornstarch, 1 part by weight of rutinand 0.5 parts by weight of riboflavin, and the resultant was tableted toobtain the captioned product, 150 mg each.

The product is a stable and easily swallowable vitamin compound ofvitamin C, vitamin P and vitamin B₂.

EXAMPLE B-7 Capsule

Ten parts by weight of calcium acetate monohydrate, 50 parts by weightof magnesium L-lactate trihydrate, 57 parts by weight of maltose, 20parts by weight of a crystalline 2-O-α-D-glucopyranosyl-L-ascorbic acidobtained by the method in Example A-2, and 12 parts by weight of aγ-cyclodextrin inclusion compound containing 20% eicosapentaenoic acidwere mixed to homogeneity, and the mixture was fed to a granulator, andthen encapsulated in gelatine to obtain capsules, 150 mg each.

The product is favorably usable as a high-quality blood cholesterollowering agent, immunopotentiator and skin-refining agent in preventiveand remedy for susceptive diseases, as well as in foodstuffs directed tothe maintenance and promotion of health.

EXAMPLE B-8 Ointment

One part by weight of sodium acetate trihydrate, 4 parts by weight ofDL-calcium lactate and 10 parts by weight of glycerine were mixed tohomogeneity, and the mixture was added to another mixture of 50 parts byweight of vaseline, 10 parts by weight of vegetable wax, 10 parts byweight of lanolin, 14.5 parts by weight of sesame oil, 1 part by weightof a crystalline 2-O-α-D-glucopyranosyl-L-ascorbic acid obtained by themethod in Example A-5 and 0.5 parts by weight of peppermint oil, andmixed to homogeneity to obtain an ointment.

The product is favorably usable as a high-quality sunscreen agent,skin-refining agent, skin-whitening agent and promoter for healinginjury and burn.

EXAMPLE B-9 Injection

A crystalline 2-O-α-D-glucopyranosyl-L-ascorbic acid, obtained by therecrystallization in accordance with the method in Experiment 1, wasdissolved in water, neutralized and sterilely filtered in usual mannerto obtain a pyrogen-free solution which was then distributed to 20 mlglass vials to give a 2-O-α-D-glucopyranosyl-L-ascorbic acid content of500 mg, dried in vacuo and sealed to obtain the captioned product.

The product is intramuscularly and intravenously administrable alone orin combination with vitamins and minerals. The product requires no coldstorage, and exhibits an excellently high solubility in saline when inuse.

Besides supplementing vitamin C, the product acts as an antioxidant toexert both activated oxygen-removing and lipoperoxideformation-inhibiting effects when hydrolyzed. Thus, the product isfavorably usable in preventive and remedy for various susceptivediseases such as viral diseases, bacterial diseases, traumatic diseases,rheumatism, immunopathies, allergy, diabetes, cataract, circulatorydiseases and malignant tumors.

EXAMPLE B-10 Injection

Six parts by weight of sodium chloride, 0.3 parts by weight of potassiumchloride, 0.2 parts by weight of calcium chloride, 3.1 parts by weightof sodium lactate, 48 parts by weight of maltose and 2 parts by weightof a crystalline 2-O-α-D-glucopyranosyl-L-ascorbic acid obtained by themethod in Example A-1 were dissolved in 1,000 parts by weight of water,and sterilely filtered in usual manner, and 250 ml aliquots of theresultant pyrogen-free solution were distributed to sterilized plasticvessels to obtain the captioned product.

The product is usable in the supplement of vitamin C, calorie andminerals. The product acts as an antioxidant to exert both activatedoxygen-removing and lipoperoxide formation-inhibiting effects whenhydrolyzed. Thus, the product is favorably usable in the restoration ofhealth during and before suffering from diseases, as well as inpreventive and remedy for susceptive diseases such as viral diseases,bacterial diseases, traumatic diseases, rheumatism, immunopathies,allergy, diabetes, cataract, circulatory diseases and malignant tumors.

EXAMPLE B-11 Intubation Nutrient

Twenty four gram aliquots of a compound consisting of 20 parts by weightof crystalline α-maltose, 1.1 parts by weight of glycine, 0.18 parts byweight of sodium glutamate, 1.2 parts by weight of sodium chloride, 1part by weight of citric acid, 0.4 parts by weight of calcium lactate,0.1 part by weight of magnesium carbonate, 0.1 part by weight of acrystalline 2-O-α-D-glucopyranosyl-L-ascorbic acid obtained by themethod in Example A-3, 0.01 part by weight of thyamine and 0.01 part byweight of riboflavin were packed in laminated aluminum bags, andheat-sealed to obtain the captioned product.

In use, one bag of the product is dissolved in about 300-500 ml ofwater, and the solution is favorably usable as an intubation nutrientdirected to oral and parenteral administration to the nasal cavity,stomach and intestine.

EXAMPLE B-12 Bath Liquid

A bath liquid was obtained by mixing 21 parts of DL-sodium lactate, 8parts by weight of sodium pyruvate, 5 parts by weight of a crystalline2-O-α-D-glucopyranosyl-L-ascorbic acid powder obtained by the method inExample A-5 and 40 parts by weight of ethanol with 26 parts by eight ofrefined water and appropriate amounts of coloring agent and flavoringagent.

The product is suitable for skin-refining agent and skin-whiteningagent, which is diluted by 100-10,000-folds in bath water when in use.In this case, bath water is replaceable with cleansing liquid,astringent and moisture liquid.

EXAMPLE B-13 Milky Lotion

One half part by weight of polyoxyethylene behenyl ether, 1 part byweight of polyoxyethylene sorbitol tetraoleate, 1 part by weight ofoil-soluble glyceryl monostearate; 0.5 parts by weight of pyruvic acid,0.5 parts by weight of behenyl alcohol, 1 part by weight of avocado oil,1 part by weight of a crystalline 2-O-α-D-glucopyranosyl-L-ascorbic acidobtained by the method in Example A-4 and appropriate amounts of vitaminE and antiseptic were dissolved in usual manner by heating, and thesolution was added with 1 part by weight of L-sodium lactate, 5 parts byweight of 1,3-butylene glycol, 0.1 part by weight of carboxyvinylpolymer and 85.3 parts by weight of refined water, emulsified with ahomogenizer, added with an appropriate amount of flavoring agent, andmixed by stirring to obtained the captioned product.

The product is favorably usable as a high-quality sunscreen agent,skin-refining agent and skin-whitening agent.

EXAMPLE B-14 Cosmetic Cream

Two parts by weight of polyoxyethylene glycol monostearate, 5 parts byweight of self-emulsifying glycerine monostearate, 2 parts by weight ofa crystalline 2-O-α-D-glucopyranosyl-L-ascorbic acid obtained by themethod in Example A-3, 1 part by weight of liquid paraffin, 10 parts byweight of glyceryl trioctanate and an appropriate amount of antisepticwere dissolved in usual manner by heating, and the mixture was addedwith 2 parts by weight of L-lactic acid, 5 parts by weight of1,3-butylene glycol and 66 parts by weight of refined water, emulsifiedwith a homogenizer, added with an appropriate amount of flavoring agent,and mixed by stirring to obtained the captioned product.

The product is favorably usable as a high-quality sunscreen cream,skin-refining agent and skin-whitening agent.

As described above, crystalline 2-O-α-D-glucopyranosyl-L-ascorbic acid,a novel substance of the invention, is substantially nonhygroscopic,free of deliquescence, consolidation and direct reducing activity,easily handleable, superiorly stable, and readily hydrolyzable in vivoto exhibit the antioxidant and physiological activities inherent toL-ascorbic acid. Furthermore, 2-O-α-D-glucopyranosyl-L-ascorbic acid isa highly safe substance because it is synthesized and metabolized invivo.

Crystalline 2-O-α-D-glucopyranosyl-L-ascorbic acid is easilycrystallizable in a supersaturated solution which is obtainable byallowing a saccharide-transferring enzyme to act alone or together withglucoamylase on a solution containing L-ascorbic acid and an α-glucosylsaccharide, and purifying and concentrating the resultant2-O-α-D-glucopyranosyl-L-ascorbic acid. Thus, such a crystallizationprocess is superior in economical efficiency, and commercializable withan ease.

Since crystalline 2-O-α-D-glucopyranosyl-L-ascorbic acid issatisfactorily high in stability and physiological activities, it isfavorably usable as a stabilizer, quality-improving agent, antioxidant,physiologically active agent, uv-absorbent pharmaceutical material andchemical in foodstuffs including beverages and processed foods,preventive and remedies for susceptive diseases, and cosmetics includingskin-refining agent and skin-whitening agent. Thus, crystalline2-O-α-D-glucopyranosyl-L-ascorbic acid has an extensive use, and is verysignificant in these industries.

We claim:
 1. A process to prepare crystalline2-O-α-D-glucopyranosyl-L-ascorbic acid, said processcomprising:providing a solution containing L-ascorbic acid and anα-glucosyl saccharide; allowing a saccharide-transferring enzyme aloneor together with glucoamylase to act on the solution to form2-O-α-D-glucopyranosyl-L-ascorbic acid; recovering and purifying theresultant 2-O-α-D-glucopyranosyl-L-ascorbic acid; preparing asupersaturated solution of 2-O-α-D-glucopyranosyl-L-ascorbic acid;crystallizing 2-O-α-D-glucopyranosyl-L-ascorbic acid in thesupersaturated solution; and recovering the resultant crystalline2-O-α-D-glucopyranosyl-L-ascorbic acid.
 2. The process of claim 1,wherein said saccharide-transferring enzyme is a member selected fromthe group consisting of cyclomaltodextrin glucanotransferase (EC2.4.1.19) and α-glucosidase.
 3. The process of claim 1, wherein saidα-glucosyl saccharide is a member selected from the group consisting ofmaltooligosaccharide, partial starch hydrolysate, liquefied starch,gelatinized starch, solubilized starch, and mixtures thereof.
 4. Theprocess of claim 1, wherein said saccharide-transferring enzyme is usedin an amount which completes the reaction within 3-80 hours.
 5. Theprocess of claim 1, wherein said saccharide-transferring enzyme isallowed to act on the solution at a pH in the range of 3-9 and atemperature in the range 20°-80° C.
 6. The process of claim 1, whereinthe concentration of the L-ascorbic acid is at least 1 w/w %.
 7. Theprocess of claim 1, wherein the concentration of the α-glucosylsaccharide is 0.5- to 30-fold higher than that of the L-ascorbic acid.8. The process of claim 1, wherein glucoamylase (EC 3.2.1.3) is allowedto act on the solution together with said saccharide-transferringenzyme.
 9. The process of claim 1, wherein said crystalline2-O-α-D-glucopyranosyl-L-ascorbic acid exhibits predominant diffractionangles (2θ) of 10.3°, 14.8°, 16.2°, 18.4° and 24.5° on powder x-raydiffraction analysis.
 10. The process of claim 1, wherein thecrystallization step is carried out in the presence of a seed crystal.11. The process of claim 10, wherein the amount of said seed crystal is0.1-10 w/w %.
 12. The process of claim 1, wherein the degree ofsupersaturation in the solution is 1.05-1.5.
 13. A foodstuff compositionwhich contains foodstuff and a crystalline2-O-α-D-glucopyranosyl-L-ascorbic acid.
 14. The foodstuff composition ofclaim 13, wherein the content of said 2-O-α-D-glucopyranosyl-L-ascorbicacid is at least 0.001 w/w %.
 15. The foodstuff composition of claim 13,wherein said crystalline 2-O-α-D-glucopyranosyl-L-ascorbic acid exhibitspredominant diffraction angles (2θ) of 10.3°, 14.8°, 16.2°, 18.4° and24.5° on powder x-ray diffraction analysis.
 16. The foodstuffcomposition of claim 13, wherein said 2-O-α-D-glucopyranosyl-L-ascorbicacid is incorporated along with a member selected from the groupconsisting of vitamin E, vitamin P, and mixture thereof.
 17. Thefoodstuff composition of claim 13, wherein said crystalline2-O-α-D-glucopyranosyl-L-acorbic acid is incorporated in said foodstuffintact or after dissolution.
 18. A cosmetic composition comprising atleast one cosmetic ingredient and further comprising a crystalline2-O-α-D-glucopyranosyl-L-ascorbic acid.
 19. The cosmetic of claim 18,wherein said crystalline 2-O-α-D-glucopyranosyl-L-ascorbic acid exhibitspredominant diffraction angles (2θ) of 10.3°, 14.8°, 16.2°, 18.4° and24.5° on powder x-ray diffraction analysis.
 20. The cosmetic of claim18, wherein said 2-O-α-D-glucopyranosyl-L-ascorbic acid is incorporatedalong with a member selected from the group consisting of vitamin E,vitamin P, and mixture thereof.
 21. The cosmetic of claim 18, whereinsaid crystalline 2-O-α-D-glucopyranosyl-L-ascorbic acid is incorporatedin a cosmetic intact or after dissolution.
 22. A method for purifying2-O-α-D-glucopyranosyl-L-ascorbic acid, said method comprising:providinga mixture containing 2-O-α-D-glucopyranosyl-L-ascorbic acid togetherwith a water-soluble contaminant; separating said mixture using awater-insoluble carrier in accordance with molecular weight or affinityinto at least two fractions; (i) a first fraction which is rich in2-O-α-D-glucopyranosyl-L-ascorbic acid, and (ii) a second fraction whichis rich in the contaminant; and recovering said first fraction.
 23. Themethod of claim 22, wherein said water-insoluble carrier is in columnform.
 24. The method of claim 22, wherein said water-soluble carrier isa strongly acidic cation exchange resin.
 25. The method of claim 24,wherein said strongly-acidic cation exchange resin is in an alkali metalor alkaline earth metal form.
 26. The method of claim 24, wherein theseparating step is carried out with gel filtration.
 27. The method ofclaim 22, wherein said mixture is obtainable by allowing asaccharide-transferring enzyme alone or together with glucoamylase toact on a solution containing L-ascorbic acid and an α-glucosylsaccharide.